1. Field of the Invention
The present invention relates generally to the field of testing samples of biological fluids for specific components therein, and, more particularly, to the quantitative determination of urea in a biological fluid such as blood based on a urease-catalyzed reaction in an organic buffer carrier solution.
2. Description of the Prior Art
The use of enzymes to aid in the determination of the concentration of various components in biological fluid is gaining rapidly as a simpler, extremely specific and more precise approach to the solution of sensing the concentration of one or more products, depletion of one or more reactants or other changes in the solution characteristics following an enzyme-catalyzed reaction in which a specific enzyme is utilized to catalyze a known reaction involving a component of interest in a fluid biological sample. One important and commonly used test involves the determination of the concentration of urea in blood serum. The level in blood serum has been found to be significant in evaluating the normality of the operation of the kidneys.
In the past a great many analytical techniques have been utilized for the analysis of biological fluids including those involving enzyme-catalyzed reactions, the results of which depend upon colorimetric techniques. These have proved to be highly susceptible to interferences or disturbances which may adversely affect the accuracy of the results. Such techniques normally rely on the action of a strong oxidizing agent or reducing agent upon the products of the enzyme-catalyzed reaction to produce a colorsensitive endpoint. These reagents, however, are generally not selective in their action on any oxidizable or reducible species and other impurities in the solution may dramatically affect the intensity of the color produced or cause a spectral shift therein which results, of course, in an inaccurate determination.
More recently, a method has been developed for the measurement of blood urea nitrogen (BUN) based on solution conductivity. It has been found that urea, which is a nonionic species, reacts with the enzyme urease to produce ionic charge carriers in an amount proportional to the original concentration of urea in the sample. The reaction may be described as follows: EQU NH.sub.2 CONH.sub.2 +H.sub.2 O .sup.urease CO.sub.3 .sup.-.sup.2 + 2NH.sub.4.sup.+
As can readily be seen from the above, the concentration of the reaction product, if sufficiently dilute, as in the biological samples tested, the hydrolysis to ammonium and carbonate ions is substantially complete and three charge carriers are produced for each molecule of urea reacted. This drastic increase in the number of charge carriers caused by the above reaction can be measured by conventional electrical techniques such electrical measurement is directly related to the concentration of urea in the biological specimen.
One prior art device which utilizes this basic principle can be found in U.S. Pat. No. 3,765,841 to Paulson et al, which illustrates and describes an apparatus for determining the concentration of, inter alia, urea in biological fluids by measuring the rate of change in conductivity of the reacting solution during a predetermined, fixed time interval in a strictly batch-type process. By that invention, a biological sample is introduced into a carrier solution along with a quantity of the enzyme urease in a free state and the rate of change of conductivity of the solution is measured after a fixed interval in which the initial portion of the reaction takes place. Because the initial stage of the reaction proceeds quite rapidly and any rate of change of conductivity measurement during the initial part of the reaction is almost infinite, the system must have an intricate built in electrical delay to allow for this initial phase to be completed prior to the measurement of the rate of change of conductivity produced by the reaction. Also, that instrument possesses all of the drawbacks inherent to a batch analyzer. Thus, after each sample is reacted and measured, both the carrier solution in which the reaction takes place and the quantity of enzyme which is utilized to catalyze the reaction are discarded. This greatly increases the cost associated with the processing of each sample.